The goal of this proposal is to clarify the means by which raphe-spinal serotonergic neurons exert antinociceptive effects. We have observed that a single system of serotonergic neurons appears to innervate both nociceptive and non-nociceptive spinal neurons. Nociceptive neurons have generally been reported to be inhibited by serotonin, whereas it has been reported that non-nociceptive neurons are excited by serotonin. Since innocuous stimuli can have antinociceptive actions, it was hypothesized that serotonergic neurons might exert their antinociceptive effects both directly, by inhibition of nociceptive neurons and indirectly, by exciting non-nociceptive neurons. This hypothesis supposes that 1) the same serotonergic neurons innervate both nociceptive and non-nociceptive neurons and 2) that the receptors for serotonin on nociceptive neurons are different from those on non-nociceptive neurons. We propose to test the first supposition by anterogradely labeling raphe-spinal serotonergic neurons and determining if the serotonergic processes innervating nociceptive neurons arise from different regions than those innervating non-nociceptive regions. In addition, we will examine the coexistence of serotonin with other neurotransmitters in varicosities opposed to nociceptive and non-nociceptive neurons, to determine if the two types of cells might be innervated by different pools of serotonergic neurons that are immunocytochemically distinguishable. The second of these suppositions will be tested by determining the types of serotonergic receptors expressed by nociceptive and non-nociceptive neurons, using in situ hybridization. It is expected that addressing these questions will clarify the means by which serotonergic neurons exert antinociceptive effects.